The neurodegenerative diseases include Alzheimer's, Parkinson's and prion disease, all of which are disorders of protein processing. Toward developing effective therapies for the protein processing diseases, we have focused on prion disease as a model system. The study of prion disease is advantageous since robust cell and transgenic (Tg) mouse models of disease are available. These cell and mouse systems facilitate measurements of disease progression and therapeutic intervention. Due to the commonality amongst the protein processing diseases, it is thought that lessons learnt in developing therapies for prion disease may have important implications for disorders like Alzheimer's and Parkinson's diseases that affect vastly greater numbers of people. Prion diseases are characterized by the misfolding of the cellular isoform of the prion protein, designated PrPc, to the disease causing isoform, denoted PrPSc. We have identified critical points in the replication of PrPSc that may be suitable for therapeutic intervention with small molecules. These include, expression and localization of the substrate, PrPc; subsequent conversion to PrPSc; and cellular clearance of PrPSc. In this application we propose to generate new screening procedures for compounds that might serve as therapeutics for prion diseases. Our goal is to develop robust, high throughput system (HTS) assays for PrPc, PrPSc accumulation, and PrPSc clearance. These in vitro screening systems will be complemented with new Tg mice that lack the multi-drug resistance (mdr) genes la and b and hence, can overcome problems traditionally associated with achieving substantial concentrations of test compounds in the CNS. Coupling the knockout of the mdrla and 1b genes with an inducible transgene system that governs PrPc expression, will allow us to measure the clearance of PrPSc as a function of high concentrations of putative therapeutics in the CNS. Besides facilitating testing of the efficacy of novel compounds, these new Tg mouse models will also aid in validating our in vitro screening targets. So as to expand the number of relevant HTS assays useful in anti-prion drug discovery, we also propose to undertake a shRNA screen to identify non-PrP genes that are involved with prion expression, replication and clearance. This study may shed light on the biology of prion disease, and importantly, lead to the development of novel HTS assays to identify selective ligands for non-PrP targets that reduce or inhibit the progression of prion disease. The development of a substantial array of HTS assays may eventually lead to an armamentarium of therapeutic compounds, which when used in concert halt the propagation of prions and hence, the progression of disease.